Monoclonal antibodies against leukocyte adhesion receptor β-chain methods of producing these antibodies and use therefore

ABSTRACT

Monoclonal antibodies and method for ameliorating an immune response disorder. The monoclonal antibodies are specific for an epitope present on the leukocyte adhesion receptor β-chain.

This is a continuation of application Ser. No. 08/200,491, filed Feb.22, 1994, now abandoned, which is a divisional application of U.S.patent application Ser. No. 08/063,063, filed May 20, 1993, nowabandoned, which is a continuation of application Ser. No. 07/917,530,filed Jul. 20, 1992, now abandoned, which is a continuation ofapplication Ser. No. 07/742,471, filed Aug. 2, 1991, now abandoned,which is a continuation of application Ser. No. 07/361,271, filed Jun.2, 1989, now abandoned.

FIELD OF THE INVENTION

This invention relates to monoclonal antibodies specific for an epitopeon the leukocyte adhesion receptor β-chain which can be used to suppressintercellular leukocyte adhesion.

DESCRIPTION OF THE BACKGROUND ART

Human immunodeficiency virus (HIV) is the etiologic agent of acquiredimmunodeficiency syndrome (AIDS), a fatal disease characterized byprofound immunosuppression, opportunistic infections, and neuropathies.Although only a small fraction of circulating lymphocytes are infectedwith the virus, there is a marked loss of T cells bearing the virusreceptor CD4. The depletion of CD4⁺ -T cells appears to contributesignificantly to the immunosuppression associated with AIDS. Syncytiumformation resulting from HIV-induced cell fusion has been shown to bethe primary cytopathic effect of the virus in vitro and has beenpostulated to account for the loss of CD4⁺ -T cells in vivo. CD4 throughits interaction with the HIV envelope glycoprotein gp120 plays animportant role in syncytium formation.

Although the CD4 receptor appears to play a significant role in theetiology of AIDS, several observations suggest that molecules on thesurface of uninfected cells other than CD4 are also involved inHIV-induced cell fusion. First, fusion of HIV-infected cells touninfected cells does not correlate with CD4-density on the surface ofthe uninfected cells. In addition, whereas transfection of non-lymphoidhuman cells with CD4 receptors renders such cells capable of fusion toHIV-infected cells, this is not true for CD4-transfected mouse cells.Finally, there is a disparity in the capacity of sera from AIDS patientsto block binding of HIV particles to CD4⁺ -cells and the capacity of thesame sera to block fusion of HIV-infected cells to CD4⁺ -uninfectedcells.

CD4 interacts directly with class II major histocompatibility complex(MHC) molecules in class II MHC-restricted T helper cell responses. Theinvolvement of the leukocyte adhesion receptor (LAR) LFA-1, in suchresponses has been demonstrated using anti-LFA-1 monoclonal antibodies(mAb). Structural similarities between gp120 and class II MHC suggestedthat the binding of gp120 to CD4 may mimic the interaction between classII MHC molecules and CD4. By analogy, the role of LAR in HIV-mediatedcell fusion was examined. In the present invention, a mAb against LFA-1completely inhibits HIV-mediated fusion of uninfected T cell blasts toHIV infected cells. This result indicates that LFA-1 is involved inHIV-induced syncytium formation, a major cytopathic mechanism of thevirus.

The LFA-1 molecule, which is expressed on T and B lymphocytes as well ason macrophages, thymocytes, granulocytes, and a subpopulation of bonemarrow cells, is composed of two non-covalently associated polypeptidesof 175,000 Kd (α; CD11a) and 95,000 Kd (β; CD18). The β-chain of LFA-1is also common to two other leukocyte antigens: Mac-1 (α-chain 165,000Kd; CD11b); the type-three complement receptor; and LeuM5 (α-chain150,000 Kd; CD11c), a molecule possibly associated with type-fourcomplement receptor activity. Although the three α-subunits differ insize, there is evidence suggesting that all three subunits are encodedby a single gene or duplicated genes. cDNA encoding the human β-chainhas been cloned and found to be 50% identical in primary structure tothe β-chain of integrin, a chick fibroblast fibronectin receptor. Thesestudies and others have shown that molecules of the LFA-1 glycoproteinfamily are members of the larger arginine-glycine-aspartate (RGD)adhesion family known as integrins.

At present, methods of limited effectiveness exist for the treatment ofAIDS or other disorders in which the intercellular interaction oflymphocytes helps to mediate the pathologic state. Those drugs which areadministered generally have severe contraindications associated withtheir use. Consequently, a considerable need exists for a therapeuticagent which can inhibit lymphocytic intercellular interaction in AIDSand other immune response mediated disorders.

SUMMARY OF THE INVENTION

One way to ameliorate immune response mediated disorders would be tosuppress intercellular leukocyte adhesion using a monoclonal antibodywhich binds to a leukocyte adhesion receptor. In so doing, intercellularleukocyte binding is suppressed thereby decreasing the likelihood ofcell-to-cell transmition of infectious agents and immune responseactivation.

In order to provide a means to ameliorate immune response mediateddisorders, the inventor has developed monoclonal antibodies which bindto an epitope on the leukocyte adhesion receptor and suppresses theability of leukocytes to adhere to each other. These monoclonalantibodies, if desired, can be therapeutically or diagnosticallylabelled.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Inhibition of syncytium formation by mAb.

FIG. 2. Dose dependent inhibition of syncytium formation by H52 IgG.

FIG. 3. H52 blocks syncytia formation at the level of PHA-blasts not 8E5cells.

FIG. 4. Inhibition of gp120 binding to CEM cells by mAb.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to monoclonal antibodies with specificityfor leukocyte adhesion receptor β-chain. These monoclonal antibodies arehighly useful for both the in vitro and in vivo immunological detectionof antigens having these β-chains and for immunotherapy of cells bearingthese receptors having these β-chains.

In a preferred embodiment of the invention a monoclonal antibody (H52)is disclosed which binds to an epitope on the leukocyte adhesionreceptor β-chain. This specificity enables H52, and like monoclonalantibodies with the specificity of H52, to be used to suppressintercellular adhesion. As a consequence, H52 is useful in amelioratingimmune response mediated disorders such as AIDS, autoimmune disease, andgraft, including graft versus host, rejection. H52 is obtained from, orhas the identifying characteristics of, an antibody obtained from thecell line having ATCC accession number HB 10160. This cell line wasplaced on deposit for 30 years at the American Type Culture Collection(ATCC) in Rockville, Md. on May 31, 1989.

Methods of Producing and Characterizing Monoclonal Antibodies

The general method used for production of hybridomas secretingmonoclonal antibodies is well known (Kohler, et al., European J. Imm.,6:292, 1976). Briefly, BALB/c mice were immunized with human splenicadherent cells and later boosted with the same type of cells. After 4days, the animals were sacrificed and the spleen cells fused with mousemyeloma P3X65 Ag8. Hybridomas were screened for antibody production andpositive clones were tested for reactivity towards human spleen tissuesections.

The present invention is directed to monoclonal antibodies, andhybridomas which produce them, which are reactive with the leukocyteadhesion receptor β-chain.

The isolation of hybridomas secreting monoclonal antibodies with thereactivity of the monoclonal antibodies of the invention can beaccomplished using routine screening techniques to determine theelementary reaction pattern of the monoclonal antibody of interest.Thus, if a monoclonal antibody being tested reacts with the leukocyteadhesion receptor β-chain such that intercellular adhesion issuppressed, then the antibody being tested and the antibody produced bythe hybridomas of the invention are equivalent.

Alternatively, it is possible to evaluate, without undueexperimentation, a monoclonal antibody to determine whether it has thesame specificity as monoclonal antibody H52 of the invention bydetermining whether the monoclonal antibody being tested prevents H52from binding to a particular antigen, for example the LFA-1 receptorwith which H52 is normally reactive. If the monoclonal antibody beingtested competes with H52, as shown by a decrease in binding by H52, thenit is likely that the two monoclonal antibodies bind to the sameepitope.

Still another way to determine whether a monoclonal antibody has thespecificity of H52 is to pre-incubate H52 with an antigen with which itis normally reactive (for example, LFA-1 receptor), and determine if themonoclonal antibody being tested is inhibited in its ability to bind theantigen. If the monoclonal antibody being tested is inhibited then, inall likelihood, it has the same epitopic specificity as the monoclonalantibody of the invention.

While the in vivo use of a monoclonal antibody from a foreign donorspecies in a different host recipient species is usually uncomplicated,a potential problem which may arise is the appearance of an adverseimmunological response by the host to antigenic determinants present onthe donor antibody. In some instances, this adverse response can be sosevere as to curtail the in vivo use of the donor antibody in the host.Further, the adverse host response may serve to hinder the intercellularadhesion-suppressing efficacy of the donor antibody. One way in which itis possible to circumvent the likelihood of an adverse immune responseoccurring in the host is by using chimeric antibodies (Sun, et al.,Hybridoma, 5 (Supplement 1):S17, 1986; Oi, et al., Bio Techniques, 4(3):214, 1986). Chimeric antibodies are antibodies in which the variousdomains of the antibodies' heavy and light chains are coded for by DNAfrom more than one species. Typically, a chimeric antibody will comprisethe variable domains of the heavy (V_(H)) and light (V_(L)) chainsderived from the donor species producing the antibody of desiredantigenic specificity, and the variable domains of the heavy (C_(H)) andlight (C_(L)) chains derived from the host recipient species. It isbelieved that by reducing the exposure of the host immune system to theantigenic determinants of the donor antibody domains, especially thosein the C_(H) region, the possibility of an adverse immunologicalresponse occurring in the recipient species will be reduced. Thus, forexample, it is possible to produce a chimeric antibody for in vivoclinical use in humans which comprises mouse V_(H) and V_(L) domainscoded for by DNA isolated from ATCC HB 10160, and C_(H) and C_(L)domains coded for with DNA isolated from a human leukocyte.

Under certain circumstances, monoclonal antibodies of one isotype mightbe more preferable than those of another in terms of their diagnostic ortherapeutic efficacy. For example, from studies on antibody-mediatedcytolysis, it is known that unmodified mouse monoclonal antibodies ofisotype gamma-2a and gamma-3 are generally more effective in lysingtarget cells than are antibodies of the gamma-1 isotype. Thisdifferential efficacy is thought to be due to the ability of thegamma-2a and gamma-3 isotypes to more actively participate in thecytolytic destruction of target cells. Particular isotypes of amonoclonal antibody can be prepared either directly, by selecting fromthe initial fusion, or prepared secondarily, from a parental hybridomasecreting a monoclonal antibody of different isotype by using the sibselection technique to isolate class-switch variants (Steplewski, etal., Proceedings of the National academy of Science, U.S.A., 82:8653,1985; Spira, et al., Journal of Immunological Methods, 74:307, 1984).Thus, the monoclonal antibodies of the invention would includeclass-switch variants having the specificity of monoclonal antibody H52which is produced by ATCC HB 10160.

When the monoclonal antibodies of the invention are used in the form offragments, such as, for example, Fab and F(ab')₂, and especially whenthese fragments are therapeutically labeled, any isotype can be usedsince amelioration of the immune response disorders in these situationsis not dependent upon complement-mediated cytolytic destruction of thosecells bearing the leukocyte adhesion receptor.

The term "immune response mediated disorder" denotes disorders in whichthe hosts' immune system contributes to the disease condition eitherdirectly or indirectly. Examples of disorders which are mediated by theimmune response includes AIDS, autoimmune disease, and graft rejection.As used herein, graft rejection encompasses both host versus graft andgraft versus host rejection.

The monoclonal antibodies of the invention can be used in any animal inwhich it is desirable to administer in vitro or in vivo immunodiagnosisor immunotherapy. The term "animal" as used herein is meant to includeboth humans as well as nonhumans.

The term "antibody" as used in this invention is meant to include intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab')₂, which are capable of binding the epitopic determinant.

Diagnostic Uses

The monoclonal antibodies of the invention are suited for use inimmunoassays in which they can be utilized in liquid phase or bound to asolid phase carrier. In addition, the monoclonal antibodies in theseimmunoassays can be detectably labeled in various ways. Examples oftypes of immunoassays which can utilize monoclonal antibodies of theinvention are competitive and noncompetitive immunoassays in either adirect or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA) and the sandwich (immunometric) assay. Detectionof the antigens using the monoclonal antibodies of the invention can bedone utilizing immunoassays which are run in either the forward,reverse, or simultaneous modes, including immunohistochemical assays onphysiological samples.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of leukocyte adhesionfactor. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses and magnetite. The natureof the carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, chemiluminescent compounds, and bioluminescentcompounds. Those of ordinary skill in the art will know of othersuitable labels for binding to the monoclonal antibody, or will be ableto ascertain such, using routine experimentation. Furthermore, thebinding of these labels to the monoclonal antibody of the invention canbe done using standard techniques common to those of ordinary skill inthe art.

For purposes of the invention, the leukocyte adhesion factor β-chainwhich is detected by the monoclonal antibodies of the invention may bepresent in biological fluids and tissues. Any sample containing adetectable amount of leukocyte adhesion factor β-chain can be used.Normally, a sample is a liquid such as urine, saliva, cerebrospinalfluid, blood, serum and the like, or a solid or semi-solid such astissues, feces, and the like.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenyl, pyridoxal, and fluorescein, which canreact with specific anti-hapten antibodies.

As used in this invention, the term "epitope" is meant to include anydeterminant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term"diagnostically effective" means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the leukocyte adhesion receptor β-chain forwhich the monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving leukocyte adhesion receptor is detectable compared to thebackground signal. Further, it is desirable that the detectably labeledmonoclonal antibody be rapidly cleared from the circulatory system inorder to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.01 mg/m² to about 20 mg/m², preferably about 0.1mg/m² to about 10 mg/m².

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobin eitherdirectly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI.

The monoclonal antibodies of the invention can be used to monitor thecourse of amelioration of an immune response mediated disorder in anindividual. Thus, by measuring the increase or decrease in the number ofleukocytes or changes in the concentration of antigen shed into variousbody fluids, it would be possible to determine whether a particulartherapeutic regimen aimed at ameliorating the immune response mediateddisorder is effective.

Therapeutic Uses

The term "ameliorate" denotes a lessening of the detrimental affect ofthe immune response mediated disorder in the animal receiving therapy.The term "therapeutically effective" means that the amount of monoclonalantibody used is of sufficient quantity to ameliorate the cause ofdisease due to the immune response.

The monoclonal antibodies of the invention can also be used forimmunotherapy in an animal having an immune response mediated disordercaused by leukocytes which express leukocyte adhesion receptor β-chainwith epitopes reactive with the monoclonal antibodies of the invention.When used in this manner, the dosage of monoclonal antibody can varyfrom about 10 mg/m² to about 2000 mg/m².

When used for immunotherapy, the monoclonal antibodies of the inventionmay be unlabeled or labeled with a therapeutic agent. These agents canbe coupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The drugs with which can be conjugated to the monoclonal antibodies ofthe invention include compounds which are classically referred to asdrugs such as for example, mitomycin C, daunorubicin, and vinblastine.

In using radioisotopically conjugated monoclonal antibodies of theinvention for immunotherapy certain isotypes may be more preferable thanothers depending on such factors as leukocyte distribution as well asisotype stability and emission. If desired, the leukocyte distributioncan be evaluated by the in vivo diagnostic techniques described above.Depending on the immune response mediated disorder some emitters may bepreferable to other. In general, alpha and beta particle-emittingradioisotopes are preferred in immunotherapy. Preferred are short range,high energy alpha emitters such as ²¹² Bi. Examples of radioisotopeswhich can be bound to the monoclonal antibodies of the invention fortherapeutic purposes are ¹²⁵ I, ¹³¹ I, ⁹⁰ Y, ⁶⁷ Cu, ²¹² Bi, ²¹¹ At, ²¹²Pb, ⁴⁷ Sc, ¹⁰⁹ Pd and ¹⁸⁸ Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is accomplished by binding thealpha-peptide chain of ricin, which is responsible for toxicity, to theantibody molecule to enable site specific delivery of the toxic effect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to aleukocyte expressing leukocyte adhesion factor β-chain for which themonoclonal antibodies of the invention are specific.

Other therapeutic agents which can be coupled to the monoclonalantibodies of the invention are known, or can be easily ascertained, bythose of ordinary skill in the art.

The dosage ranges for the administration of the monoclonal antibodies ofthe invention are those large enough to produce the desired effect inwhich the symptoms of the immune response mediated disorder areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any counterindications. Dosage can vary from about 0.1mg/m² to about 2000 mg/m², preferably about 0.1 mg/m² to about 500 mg/m²/dose, in one or more dose administrations daily, for one or severaldays. Generally, when the monoclonal antibodies of the invention areadministered conjugated with therapeutic agents lower dosages, ascompared those used for in vivo immunodiagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the monoclonal antibodies of theinvention, the medicament being used for therapy of immune responsemediated disorders due to leukocytes expressing leukocyte adhesionreceptor β-chain reactive with the monoclonal antibodies of theinvention.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLE 1 Preparation of Antiadhesion Monoclonal Factor Antibodies

Female Balb/c mice (6 to 8 weeks old) were injected intraperitoneallywith 10⁷ human splenic adherent cells in phosphate buffered saline. Thistreatment was repeated after 14 days and again after 21 days. Four daysafter the final injection the spleen was removed from one of theimmunized mice and a single cell suspension prepared. The splenic cellswere fused to Balb/c derived P3 X 653.Ag8 myeloma cells with 50%polyethylene glycol after the method of Kohler and Milstein(Nature:256:495, 1976). After establishing growing hybridoma colonies,supernatants from these cells were tested for antibodies against humanantigens by immunohistochemistry on cryostat sections (4μ) of frozenhuman spleens (Naiem, et al., J. Immuun. Meth., 50:145, 1982). H52(H52.G1.2) was selected for cloning and re-cloning byimmunohistochemistry, radioimmunoassays on human cells, and byradioimmunoprecipitation from human cells.

EXAMPLE 2 Inhibition of Syncytium Formation by Monoclonal Antibody

The effect of mAb on the fusion of 8E5 cells to PHA-blasts wasdetermined in a syncytium formation assay. The 8E5 and A3.01 cell lineswere maintained in complete medium (RPMI-1640 supplemented with 10% FBS(HyClone) and 10 mM HEPES). The 8E5 cell line is a surviving clone ofA.301 cells infected with LAV. 8E5 cells carry a single copy of theentire LAV genome but produce non-infectious virus particles due to apoint mutation in the reverse transcriptase gene. 8E5 cells express HIVenvelope glycoproteins and when mixed with CD4-positive PHA-blasts and Tcell lines produce cytopathic effects identical to those observed incultures of T cells infected with wild-type virus.

PHA-blasts were generated by incubating peripheral blood mononuclearcells for 3 days in the presence of PHA (Wellcome Diagnostics) at aconcentration of 0.25 μg/ml in complete medium. Cells were washed 3times with PBS and resuspended in complete medium at a density of 5×10⁶/ml. MAb were used in the form of purified IgG at a concentration of 25μg/ml. PHA-blasts were mixed with an equal volume (30 μl) of monoclonalantibody or medium in the wells of half-area 96-well plates (Costar) andincubated for 30 minutes at 25° C. Thirty μl of 8E5 cells were thenadded followed by incubation for 10 hr at 37° C. in a humidified CO₂incubator. Control wells consisted of PHA-blasts incubated with an equalnumber of uninfected A.301 cells. In the assay, syncytia or ballooncells consisting of 10 to 50 or more fused cells form within 4 and 10hours after mixing 8E5 cells with PHA blasts and CD4⁺ T cell lines,respectively. Continued incubation results in rapid syncytia death asdetermined by vital dye exclusion.

To determine their effect on HIV-mediated cell fusion mAb against humanleukocyte antigens were added to co-cultures of PHA-blasts and 8E5cells. The mAb tested were: H52, anti-CD18 (LFA-1 β); MHM.24, anti-CD11a(LFA-1 α); H5A4, anti-CD11b (Mac-1 α); H5A5, anti-CD45 (leukocyte commonantigen); MHM.5, anti-HLA-A,B,C; Leu3a, anti-CD4. All antibodies areIgG1,k isotype.

As shown in FIG. 1, H52, against an epitope on the β-submit of LFA-1 (CD18), completely inhibited syncytium formation. Fusion was also blockedby a mAb (MHM.24) against the α-subunit of LFA-1 (CD11a). However, themAb MHM.24 was less effective than mAb H52 since very small syncytiawere rarely observed. H5A4, a mAb against a different member of the LARfamily, Mac-1 (complement receptor type-3; CD11b), had no effect on thefusion of 8E5 cells to the PHA blasts. Also, fusion was not affected bytwo mAb recognizing unrelated cell surface proteins: MHM.5,anti-HLA-A,B,C, and H5A5, anti-leukocyte common antigen (CD45). Sincethese two antigens are expressed at equal or higher densities than LARon PHA blasts, the failure of the latter two antibodies to block fusionsuggests that inhibition by anti-LAR antibodies was not due to anon-specific steric effect. Leu3a, a mAb against CD4, which has beenpreviously shown to block binding of gp120 to CD4, completely inhibitedfusion of 8E5 to PHA-blasts (FIG. 1). Inhibition of fusion by Leu3a andthe absence of fusion between the PHA-blasts and uninfected A.301 cells(FIG. 1, control) confirmed that the fusion was mediated by HIV. Anumber of commercially available mAb against gp120 failed to inhibitfusion in the assay system. PHA-blasts and the 8E5 cells formed verylarge aggregates within 1 hr of mixing in the syncytium assay. Theseaggregates were completely inhibited by H52, MHM.24, and Leu3a, but notby the other mAb. The inhibition of syncytium formation by mAb H52 wasobserved whether the PHA blasts were mixed with 8E5 cells infected withthe mutant virus or the CEM T cell line infected with wild type HIV(HTLV-IIIB).

EXAMPLE 3 Inhibition of Syncytium Formation By H52

PHA-blasts, generated as described in Example 2, were incubated withvarious concentrations of purified H52 or PLM-2 IgG before adding 8E5cells. PLM-2 is an IgG1,k mAb against CD18 which does not inhibitLFA-1-mediated functions. The assay was carried out exactly as describedin Example 2. Syncytia were counted on an inverted microscope using alow power objective (40×) after adding trypan blue (0.1%). Data shown inFIG. 2 are the mean syncytia count/10⁶ 8E5 cells of duplicate wells.

The H52 mAb blocked 8E5-PHA-blast fusion in a dose-dependent manner withcomplete inhibition observed at concentrations above 3 μg/ml (FIG. 2).The inhibition of LFA-1-mediated lymphocyte adhesion functions by mAbH52 shows a very similar dose-dependency. PLM-2, a mAb against a CD18epitope not associated with LFA-1 adhesion functions, did not affectfusion at any concentration (FIG. 2).

Studies were also done to determine the level at which fusion wasblocked by H52. PHA-blasts and 8E5 cells (2.5×10⁶) were incubated for 1hr on ice in 0.5 ml of complete medium alone or complete mediumcontaining purified H52 or PLM-2 IgG at 25 μg/ml. After pelleting thecells, unbound mAb was removed by washing 2 times with 10 ml of PBS. Theantibody-coated PHA blasts, and 8E5 cells were then resuspended incomplete medium and mixed with uncoated 8E5 cells and PHA blasts,respectively, followed by incubation at 37° C. for 10 hr as described inExample 1. Syncytia formation was scored as described above.

Previous studies have shown that inhibition of lymphocyte interactionsby anti-LFA-1 antibodies is a uni-directional effect even when both celltypes express LFA-1. To determine if the effect of anti-LFA-1 mAb onsyncytium formation was also uni-directional, LFA-1 expression wasanalyzed by flow cytometry. Both 8E5 cells and PHA-blasts expressedLFA-1, although the expression on 8E5 was substantially less than on theblasts.

Each cell type was pre-coated with mAb H52 or the control mAb PLM-2 andwashed to remove unbound mAb before assaying syncytium formation.Pre-coating PHA-blasts with H52 resulted in near complete inhibition offusion while similar treatment of the 8E5 cells had no effect (FIG. 3).Fusion was not affected by pre-coating either the PHA-blasts or the 8E5cells with the control mAb. This result showed that the anti-LFA-1antibody blocked fusion at the level of the PHA-blast and not theHIV-infected 8E5 cells. This suggests that LAR on the CD4⁺ cellsinteracted with a ligand expressed on 8E5 cells.

EXAMPLE 4 Inhibition of HIV gp120 Binding by H52

Non-specific agents such as dextran sulfate that block the interactionof HIV envelope glycoprotein gp120 with CD4 by steric effects are knownto inhibit HIV-mediated cell--cell fusion. Consequently, the binding ofmAb H52 to LFA-1 on the surface of CD4⁺ -cells was tested to determinewhether H52 interfered with the binding of gp120 to CD4.

To purify gp120, HIV was pelleted (110,000×g, 1.5 hr) from culturesupernatants of infected PHA-blasts cells and washed once with PBS. Thevirus was resuspended in PBS and vortexed vigorously to shear off gp120,followed by centrifugation at 110,000×g. The resulting supernatant wasconcentrated using a 30,000 dalton cut-off Centricon filter. Theretained proteins, which consisted primarily of gp120 and bovine serumalbumin (BSA; 10 to 30%), were radioiodinated using the standardchloramine-T method. The labeled proteins (2 to 5 μCi/g) were diluted inPBS containing a high concentration of BSA (2%) to eliminate binding of¹²⁵ I-BSA. CD4⁺ -CEM cells (5×10⁵) were preincubated with Leu3a, H52,and PLM-2 mAb at 25 g/ml in complete medium (see Example 1) beforeadding 50 ng of radioiodinated gp120. Following a 1 hr incubation at 0°C. the cells were washed twice and bound radiolabel measured. Backgroundbinding was determined by preincubating cells with a 200-fold excess ofunlabeled gp120. Consistent with previous findings, cells pre-coatedwith the Leu3a mAb (anti-CD4) did not bind gp120 (FIG. 4). In contrast,pre-coating cells with either mAb H52 or the control mAb PLM-2 had noinhibitory effect on the binding of gp120. This result demonstrated thatinhibition of syncytium formation by mAb H52 was not due to interferencewith HIV receptor function since binding of gp120 to CD4 was not blockedby this mAb.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade without departing from the spirit or scope of the invention.

I claim:
 1. A method of ameliorating an immunological disorder in ananimal, wherein suppression of leukocyte--leukocyte cell adhesioninvolving CD18 via an epitope on the leukocyte adhesion receptor β-chainameliorates the immunological disorder comprising:administering to theanimal a therapeutically effective amount of a monoclonal antibodyproduced by ATCC HB
 10160. 2. The method of claim 1, wherein thereceptor is selected from the group consisting of LFA-1, Mac-1, and LeuM5.
 3. The method of claim 1, wherein the monoclonal antibody isadministered parenterally.
 4. The method of claim 3, wherein theparenteral administration is by subcutaneous, intramuscular,intraperitoneal, intracavity, transdermal, or intravenous injection. 5.The method of claim 1, wherein the administering is parenteral at adosage of about 0.01 mg/kg/dose to about 2000 mg/kg/dose.
 6. The methodof claim 1, wherein the monoclonal antibody is labelled with atherapeutic label.
 7. The method of claim 6, wherein the therapeuticlabel is selected from the group consisting of a radioisotope, a drug, alectin, and a toxin.